Benzoates, particularly salicylic acid (SA) and its derivatives, play critical roles in plant immune responses and basal defense through hydroxylation and glycosylation. Anthracnose is one of the most common and devastating diseases in tea plants (Camellia sinensis). However, the role of SA and its derivatives in tea plant immunity and resistance to anthracnose remains largely unexplored. In the present study, we identified and characterized a glycosyltransferase, CsUGT74B5, which was significantly downregulated in tea seedlings upon anthracnose infection. CsUGT74B5 was preferentially expressed in mature leaves and stem, and responded rapidly to exogenous SA treatment. Phylogenetic analysis suggested CsUGT74B5 might possess the catalytic activity toward benzoates. Enzymatic assays and molecular docking demonstrated recombinant CsUGT74B5 specifically glycosylated at the ortho hydroxyl groups of SA and 2, 6-dihydroxybenzoic acid (2, 6-DHBA), but did not glycosylate 2, 3-DHBA, 2, 5-DHBA, or other substrates in vitro. Overexpression of CsUGT74B5 in Arabidopsis thaliana and tobacco (Nicotiana tabacum) reduced SA level while promoting the accumulation of SA 2-O-β-D-glucoside (SAG), further validating the in vivo function of CsUGT74B5. Moreover, transient overexpression of CsUGT74B5 in two tea plant cultivars increased their sensitivity to anthracnose and accelerated lesion development, which was attributed to decreased SA levels. Overall, our finding demonstrated that CsUGT74B5-mediated biosynthesis of SAG regulated tea plant immunity against anthracnose by fine-tuning free SA levels, providing new progress into the immunity response of tea plants.

Trichomes are tiny outgrowths on the plant epidermis that serve defensive purposes against various stresses. While the regulatory mechanisms underlying unicellular trichome development are well understood, those governing multicellular trichome formation remain largely unexplored. In this study, we reveal a new regulatory pathway involving the Hair3 (H3) and H4 genes, which encode C2H2 zinc finger proteins that participate in multicellular trichome development in tomato (Solanum lycopersicum). Using CRISPR-Cas9 to generate single- and double-knockout lines, we found that h3 and h4 single-mutant plants did not show altered trichome characteristics compared to wild-type plants. However, h3/h4 double-knockout plants displayed decreased densities of Types I, VI, and VII trichomes, increased densities of Types III and V trichomes, and reduced leaf and stem lengths of Type I trichomes, revealing that H3 and H4 redundantly regulate trichome development. Notably, protein interaction assays demonstrated that H3 and H4 formed both homo- and heterodimers, supporting their cooperative role. Transcriptome and gene expression analyses identified H3 and H4 as key regulators of several genes involved in trichome development, including Woolly (Wo) and its downstream targets, such as Wox3b, MX1, H, and HD8. Protein-promoter assays showed that H3 and H4 did not directly bind to the Wo promoter but rather interacted with Wo, thereby enhancing the expression of Wo and Wox3b. These findings establish H3 and H4 as key regulators of trichome development and provide novel insights into the mechanisms controlling multicellular trichome development in tomato plants.

Plant epicuticular waxes (EW) play a critical role in defending against biotic and abiotic stresses. Notably, onions (Allium cepa L.) present a distinctive case where the mutant with defect in leaf and stalk EW showed resistance to thrips compared with the wild type with integral EW. We identified a premature stop codon mutation in the AcCER2 gene, an ortholog of CER2 gene in Arabidopsis thaliana that has been proved essential for the biosynthesis of very long-chain fatty acids (VLCFAs), in the onions with glossy leaf and stalks in our experiments. The data hinted at the possibility that this mutation might impede the elongation process of VLCFAs from C28 to C32, thereby hindering the production of 16-hentriacontanone, a primary constituent of onion EW. Transcriptomic analysis revealed substantial alterations in expression of genes in the pathways related not only to lipid synthesis and transport but also to signal transduction and cell wall modification in glossy mutants. Meanwhile, metabolomic profiling indicates a remarkable increase in flavonoid accumulation and a significant reduction in soluble sugar content in glossy mutants. These findings suggested that the enhanced resistance of glossy mutants to thrips might be a consequence of multiple physiological changes, and our integrated multiomics analysis highlighting the regulatory role of AcCER2 in these processes. Our study has yielded valuable insights into the biosynthesis of onion EW and has provided an initial hypothesis for the mechanisms underlying thrip resistance. These findings hold significant promise for the breeding programs of thrip-resistant onion.

The ubiquitin-26S proteasome system (UPS) is associated with protein stability and activity, regulation of hormone signaling, and the production of secondary metabolites in plants. Though the mechanism of action of SmMYB36 on the tanshinone and phenolic acid biosynthesis is well understood, its regulation through post-translational modifications is unclear. A constitutive photomorphogenesis 9 (COP9) signalosome subunit 5 (SmCSN5), which interacted with SmMYB36 and inhibited its ubiquitination-based degradation, was identified in Salvia miltiorrhiza. SmCSN5 promoted tanshinone biosynthesis but inhibited phenolic acid biosynthesis in the hairy roots of S. miltiorrhiza. SmMYB36 also activated the transcription of the target genes SmDXS2 and SmCPS1 but repressed that of SmRAS in a SmCSN5-dependent manner. SmCSN5 acts as a positive regulator in MeJA-induced biosynthesis of tanshinones and phenolic acids. Specifically, SmCSN5 alone, when expressed transiently in tobacco and rice protoplasts, was localized to the cytoplasm, cell membrane, and nucleus, whereas when coexpressed with SmMYB36, it was detected only in the nucleus. Additionally, the degradation of SmMYB36^1-153 by ubiquitination was lowered after truncation of the self-activating structural domain of SmMYB36^154-160. Collectively, these results suggest that SmCSN5 affected the transcriptional activation of SmMYB36 and stabilized SmMYB36, providing insights into the SmMYB36-based regulation of the accumulation of tanshinone and phenolic acid at the transcriptional and post-translational levels.

Brassinosteroids (BRs) are extensively distributed in plants and play crucial roles throughout all stages of plant growth. Nevertheless, the molecular mechanism through which BRs influence postharvest senescence in pakchoi remains elusive. Previous studies have demonstrated that the application of 1.5 μM of the BRs analog 2,4-epibrassinolide (EBR) delayed the leaf senescence in harvested pakchoi. In this study, we constructed the EBR-delayed senescence transcriptome in pakchoi leaves and discovered that EBR modulates the expression of genes involved in the chlorophyll (Chl) metabolism pathway and the BRs pathway in pakchoi. Notably, we identified and characterized an EBR-suppressed, nucleus-localized WRKY transcription factor called BrWRKY8. BrWRKY8 is a highly expressed transcriptional activator in senescent leaves, targeting the promoters of the Chl degradation-associated gene BrSGR2 and the BRs degradation-associated gene BrCHI2, thereby promoting their expression. Overexpression of the BrWRKY8 gene accelerated the senescence process in Arabidopsis leaves, while EBR treatment mitigated the leaf senescence phenotype induced by BrWRKY8 overexpression. Conversely, silencing of BrWRKY8 through the virus-induced gene silencing extended the postharvest storage period of pakchoi. In conclusion, the newly discovered BRs-BrWRKY8 regulatory model in this study provides novel insights into BRs-mediated leaf senescence in pakchoi.

Although the significance of some plant WRKYs in response to cold stress have been identified, the molecular mechanisms of most WRKYs remain unclear in grapevine. In this study, we demonstrate that cold-induced expression of VaBAM3 in Vitis amurensis executes a beneficial role in enhancing resistance by the regulating starch decomposition. VaWRKY65 was identified as an upstream transcriptional activator of VaBAM3 through yeast one-hybrid library screening and validated to directly interact with the W-box region inside the VaBAM3 promoter. Transgenic Arabidopsis thaliana plants and grapevine roots overexpression VaWRKY65 exhibited improved cold tolerance along with higher BAM activity and soluble sugar levels, whereas opposite changes were observed in VaWRKY65 knockdown lines created by virus-induced gene silencing (VIGS) in grapevine plants and in the knockout wrky65 mutants generated by CRISPR/Cas9 technology in grapevine roots. The transcriptome data show that overexpression of VaWRKY65 led to significant alteration of a diverse set of stress-related genes at the transcriptional level. One of the genes, Peroxidase 36 (VaPOD36), was further verified as a direct target of VaWRKY65. Consistently, VaWRKY65-overexpressing plants had higher VaPOD36 transcript levels and POD activity but a reduced ROS level, while silencing VaWRKY65 results in contrary changes. Collectively, these results reveal that VaWRKY65 enhanced cold tolerance through modulating soluble sugars produced from starch breakdown and ROS scavenging.

Cold stress profoundly affects the growth, development, and productivity of horticultural crops. Among the diverse strategies plants employ to mitigate the adverse effects of cold stress, flavonoids have emerged as pivotal components in enhancing plant resilience. This review was written to systematically highlight the critical role of flavonoids in plant cold tolerance, aiming to address the increasing need for sustainable horticultural practices under climate stress. We provide a comprehensive overview of the role of flavonoids in the cold tolerance of horticultural crops, emphasizing their biosynthesis pathways, molecular mechanisms, and regulatory aspects under cold stress conditions. We discuss how flavonoids act as antioxidants, scavenging reactive oxygen species (ROS) generated during cold stress, and how they regulate gene expression by modulating stress-responsive genes and pathways. Additionally, we explore the application of flavonoids in enhancing cold tolerance through genetic engineering and breeding strategies, offering insights into practical interventions for improving crop resilience. Despite significant advances, a research gap remains in understanding the precise molecular mechanisms by which specific flavonoids confer cold resistance, especially across different crop species. By addressing current knowledge gaps, proposing future research directions and highlighting implications for sustainable horticulture, we aim to advance strategies to enhance cold tolerance in horticultural crops.

The garlic bulb comprises several cloves, the swelling growth of which is significantly hindered by the accumulation of viruses. Herein, we describe a single-cell transcriptomic atlas of swelling cloves with virus accumulation, which comprised 19 681 high-quality cells representing 11 distinct cell clusters. Cells of two clusters, clusters 7 (C7) and 11 (C11), were inferred to be from the meristem. Cell trajectory analysis suggested the differentiation of clove cells to start from the meristem cells, along two pseudo-time paths. Investigation into the cell-specific activity of invasive viruses demonstrated that garlic virus genes showed relatively low-expression activity in cells of the clove meristem. There were 2060 garlic genes co-expressed with virus genes, many of which showed an association with the defense response. Five glutathione synthase/reductase genes co-expressed with virus genes displayed up-regulated expression, and the glutathione and related metabolites level showed an alteration in virus-invasive garlic clove, implying the role of glutathione in viral immunity of garlic. Our study offers valuable insights into the clove organogenesis and interaction between garlic and virus at single-cell resolution.

The CRISPR-Cas9 system can be used to introduce site-specific mutations into the genome of tomato (Solanum lycopersicum) plants. However, the direct application of this revolutionary technology to desirable tomato cultivars has been hindered by the challenges of generating transgenic plants. To address this issue, we developed an efficient and heritable genome editing system using tobacco rattle virus (TRV) for an elite tomato cultivar (the paternal line of Saladette). Notably, this virus-induced genome editing (VIGE) system enables the rapid production of various mutant seeds without the need for additional plant transformation and tissue culture, once a Cas9-expressing tomato line is established. This VIGE system consists of transgenic tomato plants that express Cas9 under the control of the tomato ubiquitin 10 (SlUbi10) gene promoter and a mobile guide RNA scaffold (gRNA:SlmFT) generated using the sequence of the tomato Flowering Locus T (SlFT) gene. We determined its editing efficiency by targeting the tomato phytoene desaturase (SlPDS) gene, which causes photobleaching symptoms when disrupted. Most transgenic seedlings infected with the TRV vectors carrying the SlPDS-targeting sgRNA developed chimeric albino leaves associated with a high frequency of indel mutations in the SlPDS gene. Remarkably, fruits from these plants yielded homozygous SlPDS knockout seeds at rates ranging from 15% to 100%. These results demonstrate the exceptional effectiveness of our VIGE system in rapidly generating heritable genome edits in tomato.

High temperatures increase the sugar concentration of grape (Vitis vinifera L.) berries, which can negatively affect the composition and quality of wine, and global climate change is expected to exacerbate this problem. Modifying the source-to-sink ratio of grapevines by selective pruning is a potential strategy to mitigate this. To investigate the effects of low source-to-sink ratio (retaining three leaves per cluster) on carbon metabolism of grape (cv. Cabernet Sauvignon) berries, we conducted an analysis of 42 metabolites and 21 enzyme activities at nine berry developmental stages，as well as transcriptomes from berries grown under two leaves per cluster. The results revealed that the metabolic pathways were coordinately regulated to maintain homeostasis under low source-to-sink ratio conditions. Because of a delay between metabolites and enzyme activities, the metabolites were loosely correlated with enzyme activities, and a lower density of connectivity between them appeared in low source-to-sink conditions. Otherwise, transcripts of the carbohydrate and amino acid metabolism pathways were enriched by carbon limitation. In summary, this integrated analysis reveals a coordinated regulation of various metabolic pathways that maintains the balance of carbon metabolism and ensures survival in challenging environments, highlighting the high metabolic plasticity of grape berries.

Salicylic acid (SA) is a phenolic phytohormone widely believed to regulate plant growth and stress response. Despite its significance, the genetic basis of SA-mediated resistance to biotic stressors in tea plants is little understood. Our study investigated the genetic diversity, population structure, and linkage disequilibrium (LD) patterns of 299 tea accessions using 79 560 high-quality single nucleotide polymorphisms (SNPs) obtained from genotyping-by-sequencing (GBS) data. Our genome-wide association study identified CSS0033791.1, an essential gene encoding 9-cis-epoxycarotenoid dioxygenase (CsNCED1), which catalyzes a vital step in abscisic acid (ABA) biosynthesis. Exogenous ABA treatment and transgenic overexpression of the CsNCED1 gene lowered SA content in the respective tea plants by inhibiting the expression of the ICS gene. Further analysis revealed that ABA could reduce the expression levels of the SA receptor gene (NPR1) and NPR1 target genes (PR1 and WRKY18), increasing the plant’s susceptibility to biotic stressors. Furthermore, the feeding behavior of Spodoptera litura revealed that the insect bite area on transgenic leaves was substantially more extensive than that in wild type (WT), implying that the CsNCED1 gene had a negative regulatory role in SA-mediated immune response. This study thus provides the foundation for future insect resistance breeding, sustainable tea plant resource usage, and molecular marker-assisted (MAS) tea plant breeding.

Fungi produce microRNA-like RNAs (milRNAs) with functional importance in various biological processes. Our previous research identified a new milRNA Foc-milR87 from Fusarium oxysporum f. sp. cubense, which contributes to fungal virulence by targeting the pathogen glycosyl hydrolase encoding gene. However, the potential roles of fungal milRNAs in interactions with hosts are not well understood. This study demonstrated that Foc-milR87 specifically suppressed the expression of MaPTI6L, a pathogenesis-related gene that encodes a transcriptional activator in the banana (Musa acuminata Cavendish group cv. ‘Baxi Jiao’) genome, by targeting the 3'untranslated region (UTR) of MaPTI6L. Transient overexpression of MaPTI6L activated plant defense responses that depend on its nuclear localization, yet co-expression with Foc-milR87 attenuated these responses. MaPTI6L enhanced plant resistance by promoting transcription of the salicylic acid signaling pathway marker gene MaEDS1. Sequence analysis of the MaPTI6L gene in 19 banana varieties, particularly those resistant to Fusarium wilt, uncovered single nucleotide polymorphisms (SNPs) at Foc-milR87 target sites. Experimental validation showed that these SNPs significantly reduce the microRNA's ability to suppress target gene expression. Our findings reveal that Foc-milR87 plays an important role in impairing plant resistance by targeting MaPTI6L mRNA and reducing MaEDS1 transcription during the early infection stage, suggesting the 3'UTR of MaPTI6L as a promising target for genome editing in generation of disease-resistant banana cultivars.

Potatoes are valued as reliable crops due to their high carbohydrate content and relatively low farming demands. Consequently, significant attention has been directed towards understanding and controlling the life cycle of potato tubers in recent years. Notably, recent studies have identified self-pruning 6A (StSP6A) as a key component of the tuberigen, the mobile signal for tuber formation, produced in leaves and then transported underground to induce tuber formation in potatoes. Recent progress in comprehending the signaling mechanisms that regulate StSP6A by photoperiod and ambient temperature components, its long-distance transport into underground tissue, and its involvement in regulating stolon tuberization has advanced significantly. Consequently, the modulation of StSP6A and other possible tuberigen signals, along with their regulatory pathways, significantly impacts potato domestication and crop yield. This progress highlights the differential regulation of tuberigen signals and their potential functions in promoting tuber formation.

Pear ring rot disease (Botryosphaeria dothidea) is a significant threat to the healthy development of the pear industry. Recent research has identified the functional role of long non-coding RNAs (lncRNAs) in various biological processes of plants. The role of lncRNAs in the pear defense response remains unknown. In this study, transcriptome sequencing was used to analyze lncRNAs in pear stem infected with B. dothidea. It identified 3555 lncRNAs, of which 286 were significantly differentially expressed. GO and KEGG analyses showed that cis- and trans-regulated target genes were enriched in multiple disease resistance-related pathways. More specifically, MSTRG.32189, predicted as an endogenous target mimic (eTM), was significantly down-regulated in response to B. dothidea infection, and was confirmed to inhibit the cleavage effect of PcmiR399b on PcUBC24. OE-MSTRG.32189 transgenic Arabidopsis exhibited lower Pi content and weaker disease resistance to Botrytis cinerea compared with wild type. In pear callus, overexpression of MSTRG.32189 negatively regulated PcmiR399b, which decreased Pi content and reduced disease resistance. Overexpressing PcmiR399b in pear callus exhibited the opposite effects compared with OE-MSTRG.32189. Overexpression and knockout of PcUBC24 further clarified that PcUBC24 negatively regulates Pi content and disease resistance to B. dothidea infection. Furthermore, the ROS levels and expressions of disease resistance pathway-related genes were regulated by the MSTRG.32189-PcmiR399b-PcUBC24 module in transgenic pear callus, which contributed to disease resistance. Overall, our results demonstrated the role of lncRNAs in the pear defense response, revealing that the MSTRG.32189-PcmiR399b-PcUBC24 module regulates phosphate accumulation and disease resistance to B. dothidea infection in pear.